CN1259068C - Dogwood extraction and its preparation method and usage - Google Patents
Dogwood extraction and its preparation method and usage Download PDFInfo
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- CN1259068C CN1259068C CN 200410014769 CN200410014769A CN1259068C CN 1259068 C CN1259068 C CN 1259068C CN 200410014769 CN200410014769 CN 200410014769 CN 200410014769 A CN200410014769 A CN 200410014769A CN 1259068 C CN1259068 C CN 1259068C
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Abstract
The present invention provides a dogwood extract, a preparation method thereof and the application thereof. The preparation method comprises: dogwood medicinal materials are extracted by alcohol, extracting solution is concentrated under reduced pressure, then water is added to the extracting solution, the mixture is precipitated, and deposits are collected and are dried under reduced pressure to obtain extract A; supernatant fluid passes through macroscopic adsorptive resin columns, impurities are removed through water washing, the gradient elution of the ethanol is carried out, and eluent is collected and is dried under reduced pressure to obtain extract B; the extract A and extract B are uniformly mixed, and the dogwood extract of the present invention is obtained. The novel pharmaceutical application of the dogwood extract of the present invention comprises preparing medicines for preventing and curing diabetic blood vessel pathological changes and medicines for lowering blood sugar.
Description
Technical field
The invention provides a kind of Fructus Corni extract, also relate to the medical usage of this preparation method of extract and this extract, belong to the field of Chinese medicines.
Background technology
Diabetes are the progressive incretion metabolism diseases of a kind of chronic whole body, and lasting hyperglycemia can cause some histoorgan Developmental and Metabolic Disorder, cause that then its dysfunction and form change.Diabetic duration surpasses 10 years, and most of patient merges the vascular complication of varying degree, especially microvascular complication.Diabetic microvascular complication is meant that microcirculation disturbance, microangioma form and the blood capillary basement membrane thickened, mainly comprises diabetic nephropathy and diabetic retinopathy, and severe patient can cause uremia or blind, has caused huge body and mind misery to the patient.Therefore, the medicine of discovery and development control diabetic microvascular complication, blood sugar lowering is significant.
Fructus Corni (Cornus officinalis Sieb et Zu cc.) has the effect of liver and kidney tonifying, essence astringing and desertion stemming, is clinical conventional Chinese medicine, China just have Fructus Corni to be applied to diabetes from ancient times (traditional Chinese medical science is called the " record of diabetes ") treatment.In recent years, Chinese scholars has been carried out many-sided research to Fructus Corni, from Fructus Corni, separate and obtain a plurality of iridoid glycoside compounds, mainly contain meliatin (loganin), morroniside (morroniside), Herba Swertiae bimaculatae glycosides (sweroside) etc. also separate having obtained ursolic acid (ursonic acid) and oleanolic acid triterpenic acid compounds such as (oleanolic acid) from Fructus Corni.Chinese patent 96109637.7 discloses Fructus Corni water decoction extract and the preparation technology thereof with immunosuppressive action.Chinese patent application 02159502.X discloses Fructus Corni water decoction extract with the effect of control ischemic vascular disease and preparation method thereof.
But up to now, Shang Weijian has the preparation technology who obtains Fructus Corni iridoid glycoside and two effective sites of total triterpene acid simultaneously, does not see that also Fructus Corni extract is used to prepare the report of control diabetic angiopathy and blood sugar lowering medicinal usage.
Summary of the invention
Technical problem to be solved by this invention is, a kind of Fructus Corni extract and preparation method thereof is provided, and the new medical use of this extract.
The technical solution adopted for the present invention to solve the technical problems is as follows.
Fructus Corni extract of the present invention is the extract that obtains by following method:
(1) Fructus Corni medical material ethanol extraction merges ethanol extract, filters the back concentrating under reduced pressure;
(2) with centrifugal behind the concentrated solution aqueous precipitation, the supernatant concentrating under reduced pressure is standby;
(3) collecting precipitation drying under reduced pressure obtains extract A after the pulverizing;
(4) middle polarity or nonpolarity macroporous adsorptive resins chromatographic column on the concentrated solution that (2) are obtained;
(5) remove impurity with the deionized water eluting after, the reuse ethanol gradient elution;
(6) collect the ethanol elution drying under reduced pressure, obtain extract B after the pulverizing;
(7) with extract A and B uniform mixing, promptly get pulverous Fructus Corni extract.
The condition and range of said extracted method is: concentration of alcohol is 10～90% in the step (1), and concentration of alcohol is 20～70% in the step (5), and extract A and B are according to the ratio uniform mixing of weight ratio 1: 3～8 in the step (7).
The optimum condition scope of said extracted method is: concentration of alcohol is 60%～90% in the step (1), and concentration of alcohol is 30～50% in the step (5).
In the said extracted method, Fructus Corni extract can adopt ethanol refluxing process, infusion process and percolation to extract.
In the Fructus Corni extract of the present invention, extract A is mainly iridoid (iridoids), and extract B is mainly triterpenic acid (triterpene acids) compounds.
The said Fructus Corni of the present invention is the mature sarcocarp of Macrocarpium plant Fructus Corni Cornus officinalis Siebet Zucc., comprises crude drug and processed product.
The present invention has carried out thin layer to Fructus Corni extract and has differentiated and assay.
(1) the thin layer discrimination condition of extract of the present invention is as follows:
1. meliatin in the extract A and morroniside thin layer are differentiated: lamellae: the silica gel G plate, developing solvent: chloroform-methanol (7: 3), developer: spray behind 10% sulphuric acid-ethanol in 105 ℃ of bakings 10 minutes, detection can be seen the speckle of meliatin and morroniside under daylight.
2. the ursolic acid thin layer in the extract B is differentiated:
Lamellae: silica gel G plate, developing solvent: petroleum ether-ethyl acetate (5: 5), developer: spray behind 10% sulphuric acid-ethanol in 105 ℃ of bakings 10 minutes, put the speckle that detection under the daylight can be seen ursolic acid; Or under ultra-violet lamp (365nm), observe fluorescence, can see the glassy yellow fluorescence of ursolic acid.
(2) extract application high performance liquid chromatogram instrument of the present invention has carried out quantitative analysis, and its assay condition is as follows:
1. the chromatographic condition of extract A:
Chromatographic column: Phenomenex
Luna 5 μ mC
18, 250 * 4.60mm; Mobile phase: methanol-water (30: 70); Flow velocity: 1ml/min; Detect wavelength: 240nm; Column temperature: 28 ℃.
By analysis, be mainly the iridoid glycoside compounds in the extract A, comprising loganin (loganin), morroniside (morroniside), Herba Swertiae bimaculatae glycosides compositions such as (sweroside), its content accounts for more than 50% of Fructus Corni extract of the present invention.
2. the chromatographic condition of extract B:
Chromatographic column: Kromasil ODS-1 (Zirchrom) 5 μ mC
18, 250 * 4.60mm; Mobile phase: acetonitrile-methanol-0.5% ammonium acetate water (69: 15: 16); Flow velocity: 1.2ml/min; Column temperature: 30 ℃.
By analysis, be mainly the triterpenic acid compounds in the extract B, comprising ursolic acid (ursonic acid) and oleanolic acid compositions such as (oleanolic acid), its content accounts for more than 3.5% of Fructus Corni extract of the present invention.
The purposes of Fructus Corni extract of the present invention is the application in preparation control diabetic angiopathy and blood sugar lowering medicine.
Beneficial effect of the present invention is as follows:
1. extraction process of the present invention is easy rationally, can obtain water solublity and fat-soluble two effective sites simultaneously.
2. the experiment proved that Fructus Corni extract of the present invention is aspect control diabetic vascular complications, the blood sugar lowering definite curative effect being arranged, especially for diabetic microvascular complications such as treatment diabetic nephropathy and diabetic renal papillary necrosis.Said diabetic microvascular complication is meant microcirculation disturbance, microangioma formation and blood capillary basement membrane thickened etc., shows as nodular glomerulosclerosis type pathological changes, diffuse mesangial sclerosis type pathological changes, and glomerular basement membrane thickening, the PE rate increases; Retina microangioma, hemorrhage, rigid ooze out or flocculence oozes out.Said blood sugar lowering is meant and reduces the blood sugar increasing that diabetes cause.
3. extract of the present invention can reduce the blood sugar increasing that diabetes cause; Significantly reduce serum proteins advanced glycation end products-peptides (AGE-P) level; Reduce the eliminating amount of microdose urine protein; Reduce the growing amount of serum early protein nonenzymatic glycosylation product-fructosamine; Improve blood vessel endothelium and nephridial tissue morphology pathological change that diabetic vascular complications causes; Suppress the formation of renal cortex AGEs, its Receptor mRNA level is descended, have the effect that alleviates the diabetic nephropathy change; Significantly reduce interior all ratios of diabetic retinal tissue in rat blood capillary, the artificial diabetes retinopathy is had certain preventive and therapeutic effect; Significantly increase the content of SOD in serum, the oxidative stress damage that is caused by diabetic vascular complications is had protective effect; The significantly content of rising diabetic vascular complications rat blood serum NO, NOS, and reduction blood plasma ET content, the dynamic equilibrium that part is recovered NO and ET, protection vascular endothelial cell; Significantly reduce the content of diabetic vascular complications rat model serum sICAM-1, TNF-α, help the generation and the development of control of diabetes vascular complication.
Experimental example 1. Fructus Corni extracts are to the therapeutical effect of chain urea assistant bacterium diabetes rat hyperglycemia
Experiment purpose: observe the therapeutical effect of extract of the present invention to chain urea assistant bacterium diabetes rat hyperglycemia
Experimental technique: get 80 of normal rats, get 10 at random as the normal control group, all the other fasting 24h, freshly prepared streptozotocin 60mg/kg causes diabetes model by rats by intraperitoneal injection, 1 week back (fasting 12h) is got tail vein and is surveyed blood glucose value with blood glucose meter, if blood glucose value 〉=16.7mmol/L and polydipsia, polyuria appear, the rat that becomes thin then classifies the diabetes model animal as.Get 36 of diabetes model rats and be divided into 3 groups at random, and model control group (normal saline 10ml/kg, ig.), positive control aminoguanidine group (0.1g.kg
-1, ig.), invention extract group (0.15g.kg
-1, ig.), other establish 10 as the normal control group (normal saline 10ml/kg, ig.), in continuous 12 weeks, respectively 6 weeks were measured fasting glucose with 12 weeks behind administration and the fasting 2h.
Experimental result: rat blood sugar obviously raises after the modeling, and the sign that polydipsia, polyuria occur and become thin.After 12 weeks of medication, except that the normal control group, each group is all seen death in various degree, and extract of the present invention and aminoguanidine all can reduce blood glucose in diabetic rats, and be good especially with extract of the present invention.The results are shown in Table 1.
Table 1 Fructus Corni extract is to the influence of chain urea assistant bacterium diabetes rat fasting glucose (X ± S)
| Group | Dosage (g.kg -1) | Number of animals (only) | Blood glucose (mmol/L) | ||
| Before the medicine | 6 weeks behind the medicine | 12 weeks behind the medicine | |||
| Normal control group model matched group extract group of the present invention aminoguanidine group | - - 0.15 0.1 | 10 8 9 8 | 5.0±1.1 21.4±2.4 △△ 21.5±2.1 △△ 21.4±2.2 △△ | 5.1±1.1 20.9±2.1 △△ 18.1±1.7 △△*## 18.5±1.9 △△*# | 5.3±1.1 20.6±1.4 △△ 16.4±0.4 △△**## 16.6±0.6 △△**## |
*P＜0.05
*Compare with model group p＜0.01; #p＜0.05 ##p＜0.01 is relatively preceding with administration; △ △ p＜0.01 with end normal matched group relatively.
Show that extract of the present invention has certain blood sugar reducing function to diabetes rat.
Experimental example 2. Fructus Corni extracts are to the influence of diabetic vascular complications rat blood serum, serum advanced glycation end products-peptide (AGE-P) level
Experiment purpose: observe the influence of extract of the present invention to diabetic vascular complications rat blood serum advanced glycation end products-peptide (AGE-P) level.
Experimental technique: get male SD rat, 180～220g, under normal feed condition, rat is earlier with 30mg.kg
-1Ip.STZ solution was pressed 0.11ml/ ip. Freund's complete adjuvant (FCA) in second day again, and 1 time weekly, continuous 3 weeks.Get tail vein after 3 weeks and survey blood glucose value, if blood glucose 〉=16.7mmol/L person then includes in and is diabetes rat with blood glucose meter.By the blood glucose value scope be divided at random model group (normal saline 10ml/kg, ig.), aminoguanidine group (0.1g.kg
-1) and extract group (0.15g.kg of the present invention
-1), (normal saline 10ml/kg, ig.), each organizes continuous ig.12 week to establish a normal group same period in addition.12 week of administration, back rat eye socket was got blood, and the utilization flowing injecting analysis technology detects the AGE-P level behind the separation of serum.
Experimental result: the diabetic vascular complications rat blood serum AGE-P level in 12 weeks of modeling significantly raises than normal group, and extract II group of the present invention and aminoguanidine group all can significantly reduce serum AGE-P level (P＜0.01), see Table 2.
The influence of table 2 pair rat diabetes vascular complication blood glucose and serum AGE-P level (X ± S)
| Group | Dosage (g.kg -1) | Number of animals (only) | Blood glucose (mmol/L) | AGE-P (mg.L -1) | |
| Before the medicine | Behind the medicine | ||||
| Normal group model group aminoguanidine group extract group of the present invention | - - 0.1 0.15 | 8 8 8 8 | 4.9±0.5 20.7±2.6 ## 20.6±2.3 ## 20.4±2.6 ## | 4.9±0.5 19.8±2.3 ## 17.7±1.5 ## 17.4±2.2 ## | 2.8±0.4 ## 14.3±3.9 ## 9.1±0.9 ##** 8.5±1.7 ##** |
*Compare with model group p＜0.01; Compare with normal group ##p＜0.01
This experimental result shows that extract group of the present invention can significantly reduce the effect of diabetic vascular complications rat model serum AGE-P content (compare with model group p＜0.01), the effect that the AGE-P level of pointing out extract of the present invention to have certain treatment diabetic vascular complications raises.
Experimental example 3. Fructus Corni extracts are to the influence of diabetic vascular complications rat blood serum fructosamine and microdose urine protein etc.
Experiment purpose: observe the effect of extract of the present invention to the diabetic vascular complications rat.
Experimental technique: get male SD rat, body weight is 180-220g, disposable celiac injection STZ (streptozotocin) 60mg.kg behind the fasting 12h
-11 week back fasting 6h gets tail vein and surveys blood glucose value with blood glucose meter, if blood glucose value 〉=16.7mmol/L person then includes in and is diabetes rat.By the blood glucose value scope be divided at random model group (normal saline 10ml/kg, ig.), aminoguanidine group (0.1g.kg
-1, ig.), extract group (0.15g kg of the present invention
-1, ig.).Establish in addition the same period one group of normal control group (normal saline 10ml/kg, ig.).Each organizes 12 weeks of continuous irrigation stomach.The 6th, 12 weeks were measured rat blood serum fructosamine, microdose urine protein etc. respectively after administration, dynamic observed the differentiation of diabetic vascular complications rat and the preventive and therapeutic effect of extract of the present invention.
Experimental result: extract of the present invention and aminoguanidine can significantly reduce fructosamine and microdose urine protein (comparing P＜0.05,0.01 with model group), the results are shown in Table 3.
Table 3 extract of the present invention is to (X ± S) such as the influence of diabetic vascular complications rat blood serum fructosamine and microdose urine protein etc.
| Group | Dosage (g.kg -1) | Number of animals (only) | Serum fructosamine (mM) | Microdose urine protein (mg.24h -1) | ||
| 6w | 12w | 6w | 12w | |||
| Normal group model group aminoguanidine group extract group of the present invention | - - 0.1 0.15 | 8 8 8 8 | 1.77±0.21 2.30±0.23 ## 1.69±0.37 ** 1.82±0.39 ** | 1.76±0.22 2.45±0.24 ## 1.95±1.45 ** 1.88±0.34 ** | 0.1±0.1 8.4±4.2 ## 3.4±3.2 * 2.6±1.6 ** | 0.1±0.1 6.9±3.7 ## 2.5±1.2 ** 2.5±1.4 ** |
##Compare with normal group P＜0.01;
*P＜0.05
*Compare with model group P＜0.01
This experiment shows that extract group of the present invention can significantly reduce the formation of diabetic vascular complications rat model fructosamine and microdose urine protein and (compare with model group, P＜0.05,0.01), point out its rising to have certain inhibitory action to diabetic vascular complications fructosamine and microdose urine protein.
Experimental example 4. Fructus Corni extracts are to diabetic vascular complications rat chest aorta blood vessel endothelium and the morphologic influence of nephridial tissue
Experiment purpose: inquire into the effect whether extract of the present invention has the control diabetic vascular complications from morphologic angle.
Experimental technique: lumbar injection STZ (streptozotocin) 60mg.kg
-11 week back fasting 6h gets tail vein and surveys blood glucose value with blood glucose meter, if blood glucose value 〉=16.7mM person then includes in and is diabetes rat.Be divided into model group, aminoguanidine group (0.1g.kg at random by the blood glucose value scope
-1, ig.), extract of the present invention (0.15g kg
-1, ig.).Establish one group of normal control group the same period in addition.Each organizes 12 weeks of continuous irrigation stomach.Experiment finishes the back and observes the thoracic aorta endothelial permeability with the method for scanning electron microscope.The form of endotheliocyte changes and leukocyte etc. is selected light microscopic for use and through corresponding dyeing (as HE and PAS) at pathological change: this paper such as arterial wall endothelium adhesions.The metamorphosis of observation glomerule such as basement membrane thickened, glycogen deposition, telangiectasis etc.: with the isostructural change of transmission electron microscope observing glomerular basement membrane.
Experimental result: each sample of model group is in morphology sees tangible pathological change, as vascular endothelial cell swelling, cell arrangement disorder and to the lumen of vessels projection, inner skin surface is uneven, the endothelium permeability increases to have obvious gaps to exist consequently between flanking cell: cell surface is coarse, sticking its surface of telling of a lot of erythrocyte, cell is arranged: glomerular volume is obviously dwindled and with diffusivity mesentery hypertrophy, glomerular basement membrane is squeezed into the striae medullares shape by normal circle button loop shape and thickens, spheroid blood capillary obturation, and sacculus enlarges relatively.Renal tubules has obvious cavity hyaline degeneration (AE degeneration).Small artery in the matter, goal arteriole glass become, thicken between kidney, and vascular endothelial cell swelling also is the cubic tube chamber that protrudes into.And the above-mentioned pathological changes of extract group of the present invention obviously alleviates, and shows effects such as substrate hypertrophy, basement membrane thickened in blood vessel endothelium protective effect preferably and the anti-glomerule.Show that extract of the present invention has the effect that improves diabetes rat morphology pathological change, we infer extract of the present invention, and caused 26S Proteasome Structure and Function change all has certain preventive and therapeutic effect to diabetic vascular complications.
Experimental example 5. Fructus Corni extracts are to the influence of diabetic vascular complications kidney of rats cortex AGEs and receptor (RAGE) mRNA expression thereof
Experiment purpose: inquire into the inhibitory action of extract of the present invention, and on molecular level, inquire into its influence to AGEs receptor (RAGE) mRNA transcriptional level in the renal cortex to diabetic vascular complications kidney of rats cortex AGEs.
Experimental technique: get male SD rat, body weight is 180-220g, disposable celiac injection STZ (streptozotocin) 60mg.kg behind the fasting 12h
-11 week back fasting 6h gets tail vein and surveys blood glucose value with blood glucose meter, if blood glucose value 〉=16.7mmol/L person then includes in and is diabetes rat.By the blood glucose value scope be divided at random model group (normal saline 10ml/kg, ig.), aminoguanidine group (0.1g.kg
-1, ig.), extract group (0.15g kg of the present invention
-1, ig.).Establish in addition the same period one group of normal control group (normal saline 10ml/kg, ig.).Each organizes 12 weeks of continuous irrigation stomach.Experiment is got right renal cortex after finishing, and has the advantages that to produce fluorescence according to AGEs, by measuring its fluorescence intensity, the content of AGEs in the secondary indication renal cortex; With the semiquantitative method of RT-PCR, amplification in vitro genes of interest RAGE, and with β-action (house-keeping gene) as confidential reference items, the expression of RAGE mRNA in the renal cortex relatively between organizing according to the ratio of both bonded EB optical density integrations.
Experimental result: model group AGEs deposition significantly increases, and RAGE expresses obviously and raises.And extract of the present invention is reducing AGEs in the renal cortex over-deposit, also can significantly suppress the high expressed of crossing of RAGE.Show that it has the effect that alleviates the diabetic nephropathy change.The results are shown in Table 4.
Table 4 extract of the present invention is to the influence of rat model renal cortex AGEs and RAGE (X ± S)
| Group | Dosage (g.kg -1) | Number of animals (only) | AGEs (AU/mg hydroxyproline) | RAGE |
| Normal group model group aminoguanidine group extract of the present invention | - - 0.1 0.15 | 10 8 9 9 | 13.8±4.6 44.3±14.5 ## 22.8±4.2 ** 21.0±5.8 ** | 0.56±0.19 2.75±1.28 ## 1.23±0.39 ** 0.83±0.17 ** |
##Compare with normal group p＜0.01;
*Compare with model group p＜0.01.
This experiment shows that extract of the present invention is reducing AGEs in the renal cortex over-deposit, also can significantly suppress the high expressed of crossing of RAGE, and shows that it has the effect that alleviates the diabetic nephropathy change.
Experimental example 6. Fructus Corni extracts are to the influence of diabetic retinal tissue in rat pathological changes
Experiment purpose: observe extract of the present invention to preventive and therapeutic effect by the inductive diabetic renal papillary necrosis of STZ.
Experimental technique: get male SD rat, body weight is 180-220g, disposable celiac injection STZ (streptozotocin) 60mg kg behind the fasting 12h
-11 week back fasting 6h gets tail vein and surveys blood glucose value with blood glucose meter, if blood glucose value 〉=16.7mmol/L person then includes in and is diabetes rat.By the blood glucose value scope be divided at random model group (normal saline 10ml/kg, ig.), aminoguanidine group (0.1g.kg
-1, ig.), extract group (0.15g kg of the present invention
-1, ig.).Establish in addition the same period one group of normal group (normal saline 10ml/kg, ig.).Each organizes 12 weeks of continuous irrigation stomach.After 12 weeks, put to death and to take out eyeball behind the rat immediately and be fixed in 4% formalin 24～48 hours.Cut eyeball along the ambitus, remove prosthomere and vitreous body, remaining eyeball is divided into 6 five equilibriums, carefully peel off retina, the flowing water flushing is after 24 hours, and with 3% pancreatin (using the Tris buffer preparation of PH7.4) digestion 3～4 hours, taking-up was put in the distilled water light towards shaking, isolate vasoganglion, be laid on the wave carrier piece and dry up naturally.PAS adds brazilwood extract dyeing, om observation.Every shop sheet is got 7 visuals field at random, counts endotheliocyte (EC) and pericyte's (PC) number and gray scale area thereof under 400 times, and calculates both ratio (E/P value), observes new vessels simultaneously and forms situation.
Experimental result: model group retinal capillary endotheliocyte number and gray scale area thereof are all apparently higher than normal rat, pericyte's number then obviously reduces, extract of the present invention and positive control aminoguanidine all can significantly reduce the interior week of diabetic retinal tissue in rat blood capillary than (P＜0.01), show that extract of the present invention has certain preventive and therapeutic effect to the artificial diabetes retinopathy.The results are shown in Table 5.
Table 5 extract of the present invention is to the influence of diabetic retinal tissue in rat pathological changes (X ± S)
| Group | Dosage (g.kg-1) | Number of animals (only) | EC (n/view) | PC (n/view) | E/P | Ec area (um2) | Pc area (um2) | E/P area |
| The normal group model group | - - | 8 9 | 21 ±5 32 ±6## | 27 ±6 12 ±5## | 0.80 ±0.22 2.94 ±1.07## | 79.9 ±24.2 135.0 ±47.6## | 82.8 ±29.4 85.1 ±39.4 | 1.08 ±0.44 1.77 ±0.71# |
| Extract group aminoguanidine group of the present invention | 0.15 0.1 | 10 10 | 24 ±5 ** 27 ±6 | 14 ±5 15 ±5 | 1.77 ±0.43 ** 1.92 ±0.29 ** | 86.1 ±23.1 ** 100.2 ±20.1 * | 55.5 ±15.5 * 65.9 ±23.3 | 1.62 ±0.43 1.59 ±0.27 |
##Compare with normal group P＜0.01;
*P＜0.05
*Compare with model group P＜0.01
This experiment shows that extract of the present invention can significantly reduce interior all ratios of diabetic retinal tissue in rat blood capillary, shows that it has certain preventive and therapeutic effect to the artificial diabetes retinopathy.
Experimental example 7. Fructus Corni extracts are to the influence of diabetic vascular complications rat model SOD in serum
Experiment purpose: by the mutation analysis to the SOD in serum value, the oxidative stress sight of tentatively inquiring into out the inductive diabetic vascular complications rat model of SFZ resembles, and the protective effect of extract of the present invention.
Experimental technique: lumbar injection STZ (streptozotocin) 60mg.kg
-11 week back fasting 6h gets tail vein and surveys blood glucose value with blood glucose meter, if blood glucose value 〉=16.7mM person then includes in and is diabetes rat.Be divided into model group, aminoguanidine group (0.1g.kg at random by the blood glucose value scope
-1, ig.), extract of the present invention (0.15g kg
-1, ig.).Establish one group of normal control group the same period in addition.Each organizes 12 weeks of continuous irrigation stomach.Measure the content of SOD in serum after the off-test with xanthine oxidase.
Table 6 extract of the present invention is to the influence of the content of rat model SOD in serum (X ± S)
| Group | Dosage (g.kg -1) | Number of animals (only) | SOD (activity/U.mL -1) |
| Normal group model group aminoguanidine group extract of the present invention | - - 0.1 0.15 | 10 8 9 8 | 50.5±5.0 38.9±4.5 ## 48.8±5.2 ** 47.5±5.4 ** |
##Compare with normal group p＜0.01;
*Compare with model group p＜0.01.
Experimental result: after 12 weeks of modeling, diabetic vascular complications rat model SOD in serum value significantly reduces than normal group; And extract of the present invention and aminoguanidine all can significantly increase the content of SOD.This experiment shows that extract of the present invention has protective effect to the oxidative stress damage that is caused by diabetic vascular complications.
Experimental example 8. Fructus Corni extracts are to the influence of diabetic vascular complications rat NO, NOS and ET
Experiment purpose: inquire into the influence of Fructus Evodiae extract surrounded by mountains, to understand its effect and mechanism of action to the diabetic vascular complications rat to NO, NOS, ET.
Experimental technique: lumbar injection STZ (streptozotocin) 60mg.kg
-11 week back fasting 6h gets tail vein and surveys blood glucose value with blood glucose meter, if blood glucose value 〉=16.7mM person then includes in and is diabetes rat.Be divided into model group, aminoguanidine group (0.1g.kg at random by the blood glucose value scope
-1, ig.), extract of the present invention (0.15g kg
-1, ig.).Establish one group of normal control group the same period in addition.Each organizes 12 weeks of continuous irrigation stomach.Experiment is got blood after finishing, and measures rat blood serum NO, NOS and blood plasma ET.
Table 7 extract of the present invention is to the influence of rat model serum NO level, NOS and blood plasma ET (X ± S)
| Group | Dosage (g.kg -1) | Number of animals (only) | NO/μmol.L -1 | NOS/U.mL -1 | ET/pg.mL -1 |
| Normal group model group aminoguanidine group extract of the present invention | - - 0.1 0.15 | 10 8 9 8 | 68.55±7.44 42.13±7.46 ## 63.55±8.12 ** 60.12±10.43 ** | 0.66±0.08 0.39±0.06 ## 0.54±0.12 * 0.53±0.10 * | 64.55±17.12 132.15±33.77 ## 78.45±18.34 ** 76.15±18.18 ** |
##Compare with normal group p＜0.01;
*P＜0.05,
*Compare with model group p＜0.01.
Experimental result: model group and normal group are relatively, NO, NOS content significantly reduce (P＜0.01, p＜0.05), ET content significantly raises (P＜0.01): and all can significantly the raise content of diabetic vascular complications rat blood serum NO, NOS of extract group of the present invention and aminoguanidine group, and reduce blood plasma ET content.This experiment shows that extract of the present invention can partly recover the dynamic equilibrium of NO and ET, and the protection vascular endothelial cell has the improvement effect to diabetic vascular complications.
Experimental example 9. Fructus Corni extracts are to the influence of diabetic vascular complications rat sICAM-1, TNF-α
Experiment purpose: inquire into the influence of extract of the present invention, analyze extract for preventing and treating diabetic vascular of the present invention to diabetic vascular complications rat blood serum soluble intercellular adhesion molecule (slCAM-1), tumor necrosis factor (TNF-α); The effect of complication and the mechanism of action thereof.
Experimental technique: lumbar injection STZ (streptozotocin) 60mg.kg
-11 week back fasting 6h gets tail vein and surveys blood glucose value with blood glucose meter, if blood glucose value 〉=16.7mM person then includes in and is diabetes rat.Be divided into model group, aminoguanidine group (0.1g.kg at random by the blood glucose value scope
-1, ig.), extract of the present invention (0.15g kg
-1, ig.).Establish one group of normal control group the same period in addition.Each organizes the continuous irrigation stomach after 12 weeks, gets serum is measured slCAM-1, TNF-α respectively with corresponding EIA Kit content.
Table 8 extract of the present invention is to the influence of rat model serum slCAM-1, TNF-α (X ± S)
| Group | Dosage (g.kg -1) | Number of animals (only) | sIAM-1(pg.ml -1) | TNF-α(pg.ml -1) |
| Normal group model group aminoguanidine group extract of the present invention | - - 0.1 0.15 | 10 8 9 8 | 0.35±0.07 0.82±0.07 ## 0.45±0.12 ** 0.46±0.09 ** | 5.5±1.2 9.7±2.5 ## 5.8±2.7 ** 5.9±1.9 ** |
##Compare with normal group p＜0.01;
*Compare with model group p＜0.01.
Experimental result: diabetic vascular complications rat model serum sICAM-1, TNF-α raise than normal group, and extract of the present invention and aminoguanidine all can significantly reduce the content of diabetic vascular complications rat model serum sICAM-1, TNF-α.This effect has generation and the development that is beneficial to the control of diabetes vascular complication.
The specific embodiment
Describe the present invention below in conjunction with embodiment.But the present invention is not limited to these given embodiment.
Embodiment 1
With Fructus Corni medical material 1000g alcohol reflux 3 times, concentration of alcohol 70%, alcohol adding amount are 15 times of medical material weight, and extraction time 120min extracts 80 ℃ of temperature.Merge extractive liquid, filters and concentrating under reduced pressure, and it is centrifugal that concentrated solution is added 4 times of water gaging post precipitations, and the collecting precipitation drying under reduced pressure obtains extract A after the pulverizing, the supernatant concentrating under reduced pressure after centrifugal is standby.
Get nonpolar macroporous adsorption resin, in adorning post with 1: 1 ratio of crude drug weight ratio, then with above-mentioned standby concentrated solution with 1 column volume/hour flow velocity by this macroporous resin column, after passing through fully, use earlier the deionized water eluting, reuse 30% and 50% ethanol carry out gradient elution, elution flow rate be 2 column volumes/hour.Collect ethanol elution, drying under reduced pressure obtains extract B after pulverizing.
Said extracted thing A and B are obtained Fructus Corni extract after according to 1: 6 ratio mixing of weight ratio, and yield is 7.02%.
Embodiment 2
With Fructus Corni medical material 1000g alcohol reflux 2 times, concentration of alcohol 85%, alcohol adding amount are 12 times of medical material weight, and extraction time 90min extracts 75 ℃ of temperature.Merge extractive liquid, filters and concentrating under reduced pressure, and it is centrifugal that concentrated solution is added 5 times of water gaging post precipitations, and the collecting precipitation drying under reduced pressure obtains extract A after the pulverizing, the supernatant concentrating under reduced pressure after centrifugal is standby.
Get the middle polarity macroporous adsorbent resin, in adorning post with 1: 2 ratio of crude drug weight ratio, then with above-mentioned standby concentrated solution with 1.5 column volumes/hour flow velocity by this macroporous resin column, after passing through fully, use earlier the deionized water eluting, reuse 50%～70% ethanol carries out gradient elution, elution flow rate be 3 column volumes/hour.Collect ethanol elution, drying under reduced pressure obtains extract B after pulverizing.
Said extracted thing A and B are obtained Fructus Corni extract after according to 1: 5 ratio mixing of weight ratio, and yield is 6.98%.
Embodiment 3
Fructus Corni medical material 1000g is pulverized the back cross 40 mesh sieves, with the percolation extraction after 48 hours of 20% alcohol dipping, flow velocity 4～5 ml/min, percolation ration is 10 times of medical material weight.Merge extractive liquid, filters and concentrating under reduced pressure, and it is centrifugal that concentrated solution is added 4 times of water gaging post precipitations, and the collecting precipitation drying under reduced pressure obtains extract A after the pulverizing, the supernatant concentrating under reduced pressure after centrifugal is standby.
Get the middle polarity macroporous adsorbent resin, in adorning post with 1: 3 ratio of crude drug weight ratio, then with above-mentioned standby concentrated solution with 1 column volume/hour flow velocity by this macroporous resin column, after passing through fully, use earlier the deionized water eluting, reuse 30%～70% ethanol carries out gradient elution, elution flow rate be 2 column volumes/hour.Collect ethanol elution, drying under reduced pressure obtains extract B after pulverizing.
Said extracted thing A and B are obtained Fructus Corni extract after according to 1: 7 ratio mixing of weight ratio, and yield is 6.01%.
Embodiment 4
Fructus Corni medical material 1000g is pulverized the back cross 30 mesh sieves, with the percolation extraction after 36 hours of 60% alcohol dipping, flow velocity 3～4 ml/min, percolation ration is 12 times of medical material weight.Merge extractive liquid, filters and concentrating under reduced pressure, and it is centrifugal that concentrated solution is added 6 times of water gaging post precipitations, and the collecting precipitation drying under reduced pressure obtains extract A after the pulverizing, the supernatant concentrating under reduced pressure after centrifugal is standby.
Get nonpolar macroporous adsorption resin, in adorning post with 1: 2 ratio of crude drug weight ratio, then with above-mentioned standby concentrated solution with 1.5 column volumes/hour flow velocity by this macroporous resin column, after passing through fully, use earlier the deionized water eluting, reuse 50%～80% ethanol carries out gradient elution, elution flow rate be 2.5 column volumes/hour.Collect ethanol elution, drying under reduced pressure obtains extract B after pulverizing.
Said extracted thing A and B are obtained Fructus Corni extract after according to 1: 4 ratio mixing of weight ratio, and yield is 5.94%.
Embodiment 5
Fructus Corni medical material 1000g is pulverized the back cross 30 mesh sieves, extract with the alcohol dipping method.Concentration of alcohol 60%, alcohol adding amount are 5 times of medical material weight, and dip time 72 hours extracts 3 times.Merge extractive liquid, filters and concentrating under reduced pressure, and it is centrifugal that concentrated solution is added 5 times of water gaging post precipitations, and the collecting precipitation drying under reduced pressure obtains extract A after the pulverizing, the supernatant concentrating under reduced pressure after centrifugal is standby.
Get the middle polarity macroporous adsorbent resin, in adorning post with 1: 1 ratio of crude drug weight ratio, then with above-mentioned standby concentrated solution with 0.5 column volume/hour flow velocity by this macroporous resin column, after passing through fully, use earlier the deionized water eluting, reuse 30%～70% ethanol carries out gradient elution, elution flow rate be 2 column volumes/hour.Collect ethanol elution, drying under reduced pressure obtains extract B after pulverizing.
Said extracted thing A and B are obtained Fructus Corni extract after according to 1: 5 ratio mixing of weight ratio, and yield is 5.79%.
Embodiment 6
Fructus Corni medical material 1000g is pulverized the back cross 20 mesh sieves, extract with the alcohol dipping method.Concentration of alcohol 50%, alcohol adding amount are 6 times of medical material weight, and dip time 96 hours extracts 2 times.Merge extractive liquid, filters and concentrating under reduced pressure, and it is centrifugal that concentrated solution is added 4 times of water gaging post precipitations, and the collecting precipitation drying under reduced pressure obtains extract A after the pulverizing, the supernatant concentrating under reduced pressure after centrifugal is standby.
Get nonpolar property macroporous adsorbent resin, in adorning post with 1: 1.5 ratio of crude drug weight ratio, then with above-mentioned standby concentrated solution with 1 column volume/hour flow velocity by this macroporous resin column, after passing through fully, use earlier the deionized water eluting, reuse 30%～50% ethanol carries out gradient elution, elution flow rate be 3 column volumes/hour.Collect ethanol elution, drying under reduced pressure obtains extract B after pulverizing.
To obtain Fructus Corni extract behind said extracted thing A and the B uniform mixing, yield is 5.67%.
Above embodiment gained Fructus Corni extract, through the high performance liquid chromatograph analysis, be mainly the iridoid glycoside compounds in the extract A, comprising loganin (loganin), morroniside (morroniside), Herba Swertiae bimaculatae glycosides compositions such as (sweroside), its content accounts for more than 50% of Fructus Corni extract of the present invention; Be mainly the triterpenic acid compounds in the extract B, comprising ursolic acid (ursonic acid) and oleanolic acid compositions such as (oleanolic acid), its content accounts for more than 3.5% of Fructus Corni extract of the present invention.
Claims (12)
1. Fructus Corni extract is characterized in that said extract is the extract that obtains by following method:
(1) Fructus Corni medical material ethanol extraction merges ethanol extract, filters the back concentrating under reduced pressure;
(2) with centrifugal behind the concentrated solution aqueous precipitation, the supernatant concentrating under reduced pressure is standby;
(3) collecting precipitation drying under reduced pressure obtains extract A after the pulverizing;
(4) middle polarity or nonpolarity macroporous adsorptive resins chromatographic column on the concentrated solution that (2) are obtained;
(5) remove impurity with the deionized water eluting after, the reuse ethanol gradient elution;
(6) collect the ethanol elution drying under reduced pressure, obtain extract B after the pulverizing;
(7) with extract A and B uniform mixing, promptly get pulverous Fructus Corni extract.
2. extract according to claim 1 is characterized in that said extract is the extract that obtains by ethanol reflux extraction.
3. extract according to claim 1 is characterized in that said extract is the extract that obtains by the ethanol percolate extraction method.
4. extract according to claim 1 is characterized in that said extract is the extract that obtains by the alcohol dipping extraction method.
5. extract according to claim 1, it is characterized in that said extract is the extract that obtains by following method: concentration of alcohol is 10～90% in the step (1), concentration of alcohol is 20～70% in the step (5), and extract A and B are according to the ratio uniform mixing of weight ratio 1: 3～8 in the step (7).
6. extract according to claim 5, it is characterized in that said extract is the extract that obtains by following method: concentration of alcohol is 60%～90% in the step (1), and concentration of alcohol is 30～50% in the step (5).
7. the preparation method of a Fructus Corni extract is characterized in that comprising the following steps:
(1) Fructus Corni medical material ethanol extraction merges ethanol extract, filters the back concentrating under reduced pressure;
(2) with centrifugal behind the concentrated solution aqueous precipitation, the supernatant concentrating under reduced pressure is standby;
(3) collecting precipitation drying under reduced pressure obtains extract A after the pulverizing;
(4) middle polarity or nonpolarity macroporous adsorptive resins chromatographic column on the concentrated solution that (2) are obtained;
(5) remove impurity with the deionized water eluting after, the reuse ethanol gradient elution;
(6) collect the ethanol elution drying under reduced pressure, obtain extract B after the pulverizing;
(7) with extract A and B uniform mixing, promptly get pulverous Fructus Corni extract.
8. method according to claim 7 is characterized in that ethanol extraction adopts reflux extraction.
9. method according to claim 7 is characterized in that ethanol extraction adopts the percolation extraction method.
10. method according to claim 7 is characterized in that ethanol extraction adopts the dipping extraction method.
11. method according to claim 7 is characterized in that: concentration of alcohol is 10～90% in the step (1), and concentration of alcohol is 20～70% in the step (5), and extract A and B are according to the ratio uniform mixing of weight ratio 1: 3～8 in the step (7).
12. method according to claim 11 is characterized in that: concentration of alcohol is 60%～90% in the step (1), and concentration of alcohol is 30～50% in the step (5).
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| CN 200410014769 CN1259068C (en) | 2004-04-28 | 2004-04-28 | Dogwood extraction and its preparation method and usage |
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| CN 200410014769 CN1259068C (en) | 2004-04-28 | 2004-04-28 | Dogwood extraction and its preparation method and usage |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US7737121B2 (en) * | 2004-07-29 | 2010-06-15 | Board Of Trustees Of Michigan State University | Insulin secretion by anthocyanins and anthocyanidins |
| US7772195B2 (en) * | 2004-07-29 | 2010-08-10 | Board Of Trustees Of Michigan State University | Methods and compositions for the treatment of obesity, insulin related diseases and hypercholesterolemia |
| CN100435810C (en) * | 2005-08-15 | 2008-11-26 | 江苏吴中苏药医药开发有限责任公司 | Dogwood fruit extract and its preparation process |
| CN101810335B (en) * | 2010-04-30 | 2012-07-25 | 西南大学 | Method for preparing health-care foods with different activities by comprehensively using dogwoods |
| CN102389162A (en) * | 2011-08-04 | 2012-03-28 | 深圳烟草工业有限责任公司 | Method for preparing cornus officinalis extract, application in cigarettes and cigarette |
| CN103054890A (en) * | 2012-12-12 | 2013-04-24 | 浙江中医药大学 | Application of cornuside in preparing pharmaceuticals used to cure diabetic nephropathy |
| CN104523809B (en) * | 2014-12-09 | 2018-08-07 | 河南中医学院 | A kind of preparation method of Fructus Corni pigment |
| CN108562682B (en) * | 2018-04-01 | 2020-10-27 | 浙江贝尼菲特药业有限公司 | Thin-layer identification method for dogwood fruit formula particles and alcohol dogwood fruit formula particles |
| CN115040558B (en) * | 2022-06-25 | 2023-05-12 | 美宝医药科技集团有限公司 | Hypoglycemic natural extract and application thereof in preparing diabetes and diabetes related metabolic syndrome products |
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